The morphology of the FGF2-dscECM-HP hydrogel was determined using SEM (X-650; Hitachi Ltd., Tokyo, Japan). The FGF2-dscECM-HP hydrogel was lyophilized using standard processing and directly observed by SEM. To observe the morphology of dscECM, the intact dscECM scaffold was lyophilized, cross-sectioned, and sputter-coated with gold for SEM observation.
X 650
Manufactured by Hitachi
Sourced in Japan
The X-650 is a high-performance laboratory equipment designed for scientific research and analysis. It is a compact and versatile instrument capable of performing a wide range of analytical tasks. The X-650 utilizes advanced technology to deliver accurate and reliable results, making it a valuable tool for researchers and technicians in various scientific fields.
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Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Normal goat serum (ngs), by Zhongshan Golden Bridge Biotechnology (1 mentions)
Ab31630, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
D max 2500, by Rigaku (1 mentions)
Mastersizer 2000, by Malvern Panalytical (1 mentions)
Fts 6000 spectrometer, by Bio-Rad (1 mentions)
18 protocols using x 650
1
SEM Analysis of FGF2-dscECM Hydrogel Morphology
2
Cell-Scaffold Construct Preparation and Imaging
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The cell-scaffold constructs were fixed with 2.5% glutaraldehyde and then osmic acid, dehydrated in a graded ethanol series and dried by critical-point drying, and then sputter-coated with gold-palladium before observation. Cell attachment was observed by SEM (X-650, Hitachi, Japan).
3
Analyzing Graft Morphology and Composition
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The cross-section and lumen surface of grafts were mounted on an aluminum foil and sputter coated with gold and palladium. Scanning electron microscope (SEM, HITACHI, X-650, Japan) at an accelerating voltage of 15 kV was used to observe the morphology of grafts structure. Based on the SEM images, fiber diameter was analyzed using Image-Pro Plus software (IPP). The pore diameter was calculated according to the method described by Dong et al.30 (link). At least six pores per image, five images per sample and three samples per group were included to obtain the calculation.
To visualize the distribution of the PCL and PDS fibers, two different fluorescent dyes were incorporated into each fibers of the graft. DiI (1 mg/mL, orange–red fluorescent) was added to PCL solution and DiO (1 mg/mL, green fluorescent) was added to PDS solution. After co-electrospinning, the PCL/PDS grafts were visualized using a laser scanning confocal microscope (CLSM; Zeiss LSM710).
To visualize the distribution of the PCL and PDS fibers, two different fluorescent dyes were incorporated into each fibers of the graft. DiI (1 mg/mL, orange–red fluorescent) was added to PCL solution and DiO (1 mg/mL, green fluorescent) was added to PDS solution. After co-electrospinning, the PCL/PDS grafts were visualized using a laser scanning confocal microscope (CLSM; Zeiss LSM710).
4
Macrophage Behavior on Scaffolding Biomaterials
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Macrophages were seeded on different membranous scaffolds (PCL, PCL-epECM/H, PCL-epECM/H-IL-4, n = 5) in 48-well plates at a density of 5 × 103 cells/well. After 1 and 3 days of culturing, the macrophages were fixed with 4% PFA (Solarbio, China) and blocked using 5% normal goat serum (Zhongshan Golden bridge Biotechnology, China) for 45 min at room temperature, and then incubated with primary antibodies for mouse anti-CD68 (1:100, Abcam, ab31630, USA), and rabbit anti-Mannose Receptor antibodies (1:200, Abcam, ab64693, USA) overnight at 4 °C. Alexa Fluor 488 goat anti-mouse IgG (1:200, Invitrogen, USA) and Alexa Fluor 546 goat anti-rabbit IgG (1:200, Invitrogen, USA) were applied to react with the primary antibodies for 2 h at room temperature. Finally, the nuclei were stained with DAPI (Southern Biotech, England) and observed with a LSCM (Leica TCS SP8, Germany). Three images were randomly selected in each sample for statistical analysis. To evaluate cell morphology (n = 5), macrophages were fixed by 2.5% glutaraldehyde and dehydrated by gradient alcohol, then were observed under a scanning electron microscope (SEM, Hitachi, X-650, Japan) at an accelerating a voltage of 15 kV. Five cells were randomly selected in each sample for statistical analysis.
5
Thermo-Responsive Chitosan Hydrogel Synthesis
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According to the previous literature, we prepared CS hydrogel [29 (link), 30 (link)]. Thermo-responsive chitosan hydrogel was tested under different temperature conditions. The chitosan powder was dissolved in 0.1 M acetic acid, sterilized through a 0.22-μm filter, prepared into a 2% chitosan stock solution, and stored at 4 °C. The 50% β-glycerophosphate (β-GP) solution was added to the CS solution at a volume ratio of 5:1 and stirred continuously in an ice bath until the two solutions were completely mixed. After incubating at 37 °C for 30 min, the CS solution could cross-link into the hydrogel. CS hydrogel was freeze-dried under vacuum for 2 days, and the surface morphology and void size were observed under a scanning electron microscope (SEM; HITACHI X-650, Tokyo, Japan) after gold spraying.
6
Scanning Electron Microscopy of S. aureus Membrane
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To assess the alteration of S. aureus membrane morphology and ultrastructure, SEM was performed as described previously (Jan, et al., 2019 ). The bacterial suspension was transferred on glass coverslips and fixed in fresh 2.5 % glutaraldehyde at 4°C overnight. Subsequently, the coverslips were washed with PBS and incubated with 1 % osmium tetroxide at 4°C for 2 h. Following ethanol dehydration, the samples were freeze‐dried, coated with gold and observed using a SEM (X650, Hitachi; Tokyo, Japan).
7
Scanning Electron Microscopy of C. sakazakii
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SEM was performed as described previously (Jan et al., 2019 (link)). In brief, C. sakazakii cells were collected, added to the fixative buffer for 2 h at room temperature, and then fixed at 4°C overnight. The sample was then sequentially washed 3 times and eluted with 50, 70, 80, and 90% ethanol, respectively. After another round of centrifugation, ethanol was added to the pellet and incubated for 30 min. Upon ethanol dehydration, the samples were then freeze-dried, coated with gold and observed using the scanning electron microscope (X650, Hitachi, Tokyo, Japan).
8
Characterization of Crystalline Materials
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The powder X-ray diffraction (PXRD) patterns were obtained on a Rigaku D/max-2500 (Rigaku, Japan) using Cu-Kα radiation (λ = 1.5405 Å), with a step size of 0.02° and scanning rate of 8° per min over diffraction angle (2θ) range of 5°–40°. The test temperature was 298 K, the measured voltage was 40 kV, and the current was 15 mA. Crystal shapes were observed by field-emission scanning electron microscopy (SEM, Hitachi X-650). Differential scanning calorimetry (DSC) was performed from 303 to 570 K on a Mettler-Toledo 1/500 calorimeter. Thermogravimetric analysis (TGA) was carried out from 303 to 600 K on a Mettler-Toledo 1/SF analyzer at a constant heating rate of 10 K min−1 under a nitrogen gas flow rate of 50 mL min−1.
9
Gonad and Sperm Analysis of 2nNCRC Hybrid
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One female 2nNCRC individual was randomly sampled (only six living 2nNCRC-F1 of 2nCOC (♀) × 2nBSB (♂) were observed in 2014; four females and two males). The 2nNCRC gonad was fixed in Bouin’s solution for the preparation of tissue sections. Paraffin-embedded sections were cut and stained with haematoxylin and eosin. The gonadal structure was observed under a light microscope and photographed with a Pixera Pro 600ES (Pixera Corporation, Santa Clara, CA, USA).
2nNCRC, 2nGCOC, and 2nGMCC semen samples were collected with a clean sucker and transferred into a 2.5% glutaraldehyde solution. The samples were then centrifuged at 2000 × g for 1 minute and fixed in a 4% glutaraldehyde solution overnight and in a 1% osmic acid solution for 2 hours. The spermatozoa were dehydrated in alcohol, added dropwise onto slides, desiccated, subjected to atomized gilding and analysed with an X-650 (Hitachi) SEM scanning electron microscope (Nikon, Japan).
2nNCRC, 2nGCOC, and 2nGMCC semen samples were collected with a clean sucker and transferred into a 2.5% glutaraldehyde solution. The samples were then centrifuged at 2000 × g for 1 minute and fixed in a 4% glutaraldehyde solution overnight and in a 1% osmic acid solution for 2 hours. The spermatozoa were dehydrated in alcohol, added dropwise onto slides, desiccated, subjected to atomized gilding and analysed with an X-650 (Hitachi) SEM scanning electron microscope (Nikon, Japan).
10
Characterization of CS/HA Nanoparticles
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Nanoparticles were prepared in ultrapure water at 25 °C. The average particle size and zeta potential of nanoparticles were measured by a Mastersizer 2000 laser diffractometer (Malvern Instruments, Worcestershire, UK). Each experiment was repeated three times. Prior to Scanning Electron Microscopy (SEM) analysis the CS/HA nanoparticles in solutions were carefully dropped onto a silica surface and were lyophilized overnight, then were sputter coated with a layer of gold. Micrograph was observed using SEM (Hitachi X-650, Japan).
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